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静宁鸡肌肉发育调控机理
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摘要
静宁鸡属于甘肃省特有的地方鸡种,肉蛋兼用,目前已经处于濒临灭绝的状况。静宁鸡与肉鸡具有相同的起源,具有大体相同的遗传背景,但也存在与其特定性状如肌肉生长发育相关的差异。本论文以静宁鸡鸡胚为主要研究对象,选择生长发育相关蛋白:猫眼综合症候选基因2(CECR2)、胰岛素样生长因子1(IGF-1);脂肪酸代谢相关蛋白:细胞外脂肪酸结合蛋白(Ex-FABP)、脂蛋白脂酶(LPL);蛋白质降解相关蛋白:钙蛋白酶抑制蛋白(CAST)、钙蛋白酶1(CAPN1)。用免疫组织化学方法观察鸡胚发育HH19-31期后肢芽和体节中以上蛋白的表达分布,Western blotting方法检测静宁鸡鸡胚在HH19、HH24、HH27、HH31各期及孵化13天(E13)、孵化18天(E18)后肢肌肉中以上蛋白的表达水平变化,并与白羽肉鸡、黄羽肉鸡、海兰褐蛋鸡鸡胚中的表达进行比较,旨在了解静宁鸡肌肉生长发育规律,探寻不同发育期参与静宁鸡鸡胚肌肉发育调控的蛋白。结果如下:1行为学、组织学与扫描电镜观察
     体视显微镜下观察鸡胚各时期的发育特征与Hamburger的分期特征基本一致,可以按期进行取材。从HH19期到HH31期,不同品系鸡胚5min内运动行为有差异,黄羽肉鸡和白羽肉鸡的运动要普遍高于静宁鸡和海兰蛋鸡。鸡胚重量随着孵化天数的增加而增长,但增重程度不一致。成年静宁鸡在肉质特性方面优于黄羽肉鸡。提示不同品种选择的结果可能使得细胞在早期增殖、分化水平上已经发生改变,不同品种间生长速度的差异在胚胎期就表现出来了。2生长发育相关蛋白在静宁鸡鸡胚发育中的表达
     2.1免疫组化结果发现,CECR2蛋白在不同品系鸡胚发育中的肢芽、体节都有表达。HH19期CECR2在靠近喙端的体节、肢芽边缘区表达;HH21-27期:CECR2表达延伸至尾端体节、肢芽边缘区;HH29-31期:CECR2在体节、肢芽中还有表达。Western blotting结果:白羽肉鸡和黄羽肉鸡肌肉中CECR2蛋白在整个过程一直较高表达,静宁鸡肢芽发育HH19-24期CECR2蛋白几乎不表达,在HH27期时表达突然升高,到13天时已经超过了黄羽肉鸡和白羽肉鸡,而蛋鸡表达变化不大。这表明CECR2蛋白参与了静宁鸡早期肌节发育和肌细胞发生,在不同种鸡各个发育时期出现的时间以及表达的情况都不一致,这可能与不同的肌细胞发育有关。
     2.2免疫组化结果发现,IGF-1蛋白在不同品系鸡胚中都有表达,细胞内表达部位主要为细胞质。HH19期:IGF-1蛋白在外胚层侧板有表达;HH21-27期:IGF-1在肢芽外胚层顶外胚层嵴、非顶外胚层嵴区、软骨形成区有分布。HH31期:在前软骨雏形区,软骨膜周围,细胞坏死区也出现。Western blotting结果:在静宁鸡肌肉发育过程中,IGF-1蛋白表达水平持续增加,其中在HH27-31期间表达水平最高,从HH31期开始又出现降低,显著低于白羽肉鸡,与黄羽肉鸡的表达相当。以上结果表明肌肉IGF-1蛋白表达存在品系差异,在鸡胚发育中肌原细胞程序性的激活和肌肉特异基因的表达受到IGF-1的影响,IGF-1可能参与调控静宁鸡胚胎肌肉发生中成肌细胞的增殖和肌管融合。
     3脂肪酸代谢相关蛋白在静宁鸡鸡胚发育中的表达
     3.1免疫组织化学结果表明,在鸡胚早期发育中,Ex-FABP蛋白在整个肢芽都有分布。在早期,Ex-FABP蛋白在间质细胞中有表达,HH29期时,血管上也有分布。HH31期时,肌细胞上Ex-FABP蛋白表达增多,间质细胞中表达减少。Western blotting结果:静宁鸡在整个过程中Ex-FABP蛋白表达水平均较高,高于同期的肉鸡和蛋鸡。结果表明在胚胎发育初期,静宁鸡体内Ex-FABP蛋白的表达与早期肌肉发育有关系,可能会促进肌肉肌间脂肪的沉积,也可能会作为能量的提供者,在静宁鸡耐寒性状中发挥作用。
     3.2免疫组化结果发现,LPL蛋白在鸡胚发育中的肢芽和体节均有表达。HH19-31期:LPL蛋白在肢芽外胚层顶外胚层嵴、非顶外胚层嵴区、软骨形成区有分布,脂肪细胞中也有分布。Western blotting结果:在发育的各个时期,静宁鸡LPL蛋白表达一直处于较高的水平,在HH27、31、E18期,表达量最高,与黄羽肉鸡和白羽肉鸡有显著性差异。静宁鸡胚胎肢芽肌肉组织发育过程中LPL的表达总体较高,这一时期主要是器官形成和分化期,可能LPL并不是静宁鸡体内调节肌肉发育的主要因子,它可能更多地与肌细胞的能量需求及肌内脂肪沉积相关,从而提高其在恶劣环境下的生存能力。表达时间上的差异可能是由于静宁鸡LPL基因表达启动早于其它鸡,对静宁鸡早期胚胎发育能量供应效应要高于其它鸡。
     4蛋白质降解相关蛋白在静宁鸡鸡胚发育中的表达
     4.1免疫组化结果发现,从HH19-31期,CAST蛋白在肢芽和体节中广泛分布。Western blotting结果:在HH19-24期,静宁鸡CAST蛋白的表达均高于其它三个品系,随后静宁鸡表达下降;第13-18天,静宁鸡表达又升高,与黄羽肉鸡、白羽肉鸡、海兰蛋鸡间差异显著。结果表明钙蛋白酶抑制酶在鸡细胞内是普遍存在的,由于肝脏为优势表达组织,而胚胎期肌肉组织表达不占优势,因而此基因在静宁鸡早期腿肌发育中不是主要的调控因子,可能更多的与嫩度相关。
     4.2免疫组化研究发现,CAPN1蛋白在鸡胚肢芽发育中均有表达,但是表达量相对较弱。Western blotting蛋白定量:在发育的各个时期,静宁鸡CAPN1蛋白表达一直比较稳定,其中在HH27-HH31期之间表达较高。HH19期到13天之间,静宁鸡CAPN1蛋白表达水平略低于黄羽肉鸡和白羽肉鸡,但高于海兰蛋鸡;到第18天时,静宁鸡CAPN1表达水平略低于白羽肉鸡,高于黄羽肉鸡,和海兰蛋鸡间有显著性差异(p<0.05)。结果表明CAPN1参与了早期鸡胚肌肉发育。在静宁鸡发育早期中CAPN1蛋白表达水平的变化,可能是满足肌细胞生长发育中对蛋白质新陈代谢的需求;发育后期第13天到第18天中又出现降低,可能是与肉质嫩度有关系。
     总之,在静宁鸡鸡胚发育中,猫眼综合症候选基因2、胰岛素样生长因子1、细胞外脂肪酸结合蛋白、脂蛋白脂酶、钙蛋白酶抑制蛋白、钙蛋白酶1参与了肌肉发育调控。静宁鸡与白羽肉鸡、黄羽肉鸡、海兰褐蛋鸡在肌肉发育中存在差异,可能是由于不同蛋白对肌肉发育调节能力不同所致。
Jingning chicken is a special local breed for combination of meat and eggs in Gansu province. Now they are already in endanger. Jingning chicken and broiler chicken, with the same origin, have the same genetic background, but there are also differences that related to their specific properties such as muscle growth and development. This study was carried out on protein expression of different periods of Jingning chicken embryos, being investigted such as the cat's eye syndrome candidate gene2(CECR2), insulin-like growth factor1(IGF-1), extracellular fatty acid binding protein (Ex-FABP), lipoprotein lipase (LPL), calpastatin(CAST) and calpainl(CAPN1) by using immunohistochemistry method. The expression level of above protein were detected by Western blotting, compared with the expression in embryos of Baiyu broilers, Huangyu broilers and hy-line brown layers. The results are as follows:
     1Behavioral and histological observation
     Characteristics of different periods in stereo microscope were according to Mr. Hamburger's, therefor the chicken embryo could be get on schedule. Different strains of chicken embryo had motion difference in five minutes from HH19to HH31period. Huangyu broilers and Baiyu broilers had all higher movements than Jingning chicken and hy-line brown layers. The weight of embryos increased with the incubation days. Adult Jingning chicken had better meat properties than the other breeds. The results indicated that species selection may lead to cells proliferation and differentiation at early stages. The different growth rate among breeds were already show in embryonic stages.
     2Expression of factors related to muscle growth in Jingning chicken embryos
     2.1Immunohistochemistry results showed that CECR2protein was expressed in limb bud and somite of different chicken embryos. At HH19period, CECR2protein was expressed near the beak to the somite and fringes of limb bud. From HH21to27period,CECR2protein expression was extend to the rear somite and fringes of limb bud. From HH29to31period, CECR2expression was also seen in the somite and limb bud. Western blotting results showed that Huangyu broilers and Baiyu broilers had higher CECR2expression throughout the entire period, Jingning chicken hardly express CECR2protein from HH19to HH24, but there was a sudden increase in HH27, and to E13days beyond Huangyu broilers and Baiyu broilers. Hy-line brown layers didn't show much. The results suggested that CECR2protein was involved in early muscle cells growth of Jingning chicken. In each developmental period, different chicken breeds had differ expression, this may be related to different muscle cell development.
     2.2Immunohistochemistry results showed IGF-1protein were expressed in different strains of chicken embryos. At HH19period, IGF-1protein was expressed at the non-AER section, AER, and cartilage formation area. At HH31period, IGF-1protein was expressed at the cartilage prototype area, perichondrium and cell necrosis area. Western blotting results showed that IGF-1expression level in Jingning chicken continued to increase and have the highest expression level during HH27to HH31. From HH31to E18, IGF-1expression level in Jingning chicken is lower than in Huangyu broilers and Baiyu broilers. The results showed that IGF-1protein expression have strain differences in muscle, programmed cell activation and muscle specific gene expression in chicken embryos were affected by IGF-1.Therefor, IGF-1may participate in muscle cell proliferation and myotube fusion during Jingning chicken embryo stages.
     3Expression of factors involved in fatty acid metabolism in Jingning chicken embryos
     3.1Immunohistochemistry results showed that in the early development of embryos, the Ex-FABP was distributed in the entire limb bud. At early stage, mesenchymal cells showed Ex-FABPprotein expression. Ex-FABP protein were observed also in the large blood vessels. At HH31, the protein became prominent in myofibers and also found in mesenchymal cells. Western blotting results showed in the whole process, that the Ex-FABP protein expression levels of Jingning chicken were higher than broilers and laying hens at the same period. Results showed that during the early embryonic development, the Ex-FABP protein expression in Jingning chicken may promote muscle development as well as muscle fat deposition, even as a source of fat decomposition to supply energy for Jingning chicken during cold environment.
     3.2Immunohistochemistry results showed that LPL protein were expressed in developing limb bud and segment. Western blotting results were that Jingning chicken had higher LPL protein expression during the period, especially at HH27, HH31and E18. Jingning chicken had the highest LPL protein expression. The results showed that higher LPL expression in Jingning chicken may be according to the quicker organ formation and differentiation stage, it may do more to relate with energy demand and intramuscular fat deposition. Time differences during expression may be due to earlier LPL gene expression in Jingning chickens than in other chickens.Therefor, supply more energy for Jingning chickens during early embryonic development.
     4Expression of factors involved in protein degradation in Jingning chicken embryos
     4.1Immunohistochemistry results showed that from HH19to HH31, CAST were widely distributed in the limb bud and segment. Western blotting results showd that, from HH19to HH24, CAST protein expression of Jingning chicken were higher than the other three strains and then declined. From E13to E18, the expression of Jingning chicken rose again, and had significant difference with the other three breeds (p<0.05). The results showed that calpastatin was ubiquitous in chicken cells, this may be due to the liver to be an advantage organ for CAST expression.And then, the protein expression in early Jingning chicken limb muscle development is not the main regulatory factors.
     4.2Immunohistochemistry results showed that CAPN1protein were expressed in chicken embryo limb bud during development periods, but relatively weak. Western blotting results showed that CAPN1protein expression of JingNing chicken has been relatively stable. From HH19to E13, CAPN1JingNing chicken expression level was slightly lower than Huangyu broilers and Baiyu broilers, but higher than layers. Upto E18, CAPN1expression level in Jingning chicken embryo was slightly lower than Baiyu broilers and higher than Huangyu broilers. The results showed that CAPN1was involved in the early embryo development. Changes of CAPN1protein expression in Jingning chicken development level may be meet to demand for protein metabolism in muscle cell growth and meat tenderness.
     In conclusion, cat's eye syndrome candidate gene2, insulin-like growth factor1, extracellular fatty acid binding protein, lipoprotein lipase, calpastatin and calpain1were involved in muscle development during different stages of Jingning chicken embryos. Jingning chicken had difference with Baiyu broilers, Huangyu broilers and hy-line brown layers in the muscle characteristic, this may be due to different regulation ability by above protein on muscle development.
引文
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